Our overall goal is to optimize hypotonic rectal enema formulations for delivering tenofovir (TFV) and TFV analogs to the entire colorectal surface with as high a dose as can be delivered safely. We have investigated the role of enema vehicle tonicity in drug delivery in the mouse rectum, including hypotonic formulations that cause rapid water uptake from the lumen, and hypertonic formulations that cause rapid secretion of water into the lumen. Our results are consistent with the studies done by Hendrix and coworkers in the human colorectum: (i) tap water, a maximally hypotonic formulation, drives drug delivery and absorption locally, and not as far up the colon as is likely necessary, and (ii) Fleet enema, a markedly hypertonic vehicle, causes fluid secretion that prevents uniform drug distribution. In contrast, moderately hypotonic formulations provide the best surface distribution, and they do so rapidly. Hypotonic formulations deliver drugs and drug-loaded nanocrystals by advection caused by sodium (Na+)-driven, osmotically-induced flow of water from the lumen into the tissues, similar to the natural process by which the colorectum dehydrates the feces. Importantly, relatively high Na+ in semen makes it hypotonic in the colorectum, which is likely to deliver HIV rapidly to the epithelium by advection, thus suggesting that our moderately-hypotonic enema drug delivery method may be critical to ensure that drug is delivered to all surfaces where HIV can reach. In Aim 1, we will optimize a mildly hypotonic formulation for delivering TFV and TFV analogs to the colorectum of mice, and then in non-human primates (NHP), to select the enhanced bioavailability product to be used in clinical studies in Projects 1 and 4. In Aim 2, we will be designing an enhanced enema product for increased bioavailability, and sustained drug release. In Aim 2A, we will determine whether mucus-penetrating drug nanocrystals can prolong the duration of prophylactic drug concentrations of TFV and TFV analogs in mice, and then in NHP. In Aim 2B, we will test the hypothesis that a gelling agent will prolong retention on the colorectal surface, and possibly form a barrier to HIV. In Aim 2C, we will test the product with the optimized combination of bioavailability enhancements and sustained release properties for protection against SHIV challenge in NHP.